Sensitized cultured cells for botulinum toxin characterization

ABSTRACT

Apparatus, systems and methods can provide improved detection of botulinum neurotoxins. In one aspect an isoquinolynyl compound can be used to enhance the sensitivity of both Förster resonance energy transfer (FRET) and non-FRET cell-based assays. In another aspect, non-FRET assays and constructs utilize a reporter that is not coupled with the second fluorophore in a manner that produces significant FRET. In that subject matter an environment cell can include an enzyme that facilitates degradation of the reporter significantly faster after the cleavage than before the cleavage, and presence of the Botulinum toxin correlates with reduction of the signal from a baseline signal. Where the environment is a cell, the cell can advantageously express both the construct that includes the reporter, and an enzyme that facilitates the degradation.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.13/733,803, filed Jan. 3, 2013, issued as U.S. Pat. No. 9,303,285, whichclaims priority to U.S. Provisional Patent Application No. 61/582940,filed Jan. 4, 2012 and is a continuation in part of U.S. patentapplication Ser. No. 13/485537, filed May 31, 2012, and issued as U.S.Pat. No. 9,274,121, which claims the benefit of U.S. ProvisionalApplication No. 61/492,237, filed Jun. 1, 2011, the disclosures of whichare incorporated herein by reference in their entirety. These and allother extrinsic materials discussed herein are incorporated by referencein their entirety. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

FIELD OF THE INVENTION

The field of the invention is characterization of Botulinum Neurotoxins(BoNTs).

BACKGROUND

Botulinum Neurotoxins are the most lethal substances known, anddepending on the scrotype, the estimated human lethal dose ranges from 1to 3 ng toxin per kg body weight. Due to their ease of purification andhigh potency, BoNTs pose a real and potential threat for use asbioweapons. Further, BoNTs are increasing used for cosmetic andtherapeutic purposes. Currently, the majority of BoNT detection andpotency measurements completed in government and industrial laboratoriesare done using animal-based assays that suffer from high costs and pooraccuracy.

The primary advantage of using cell-based assays (CBAs) for BoNT potencymeasurements is that CBA systems closely mimic the individualphysiologic steps that occur in neurons during intoxication. BoNTs arezinc-dependent endopeptidases composed of a heavy chain, responsible forneuron-specific receptor binding and cell entry, and a catalytic lightchain responsible for synaptic protein cleavage. Upon entry intoneurons, the BoNTs specifically disrupt the protein machineryresponsible for fusion of synaptic vesicles with the plasma membrane,thereby inhibiting neurotransmitter release into the post-synapticjunction. Because all physiologic steps must be accounted for in CBAs,most assays fail to meet the sensitivity requirements of BoNT activitymeasurement.

The employment of established, stable model cell lines for the detectionof BoNTs have recently been described, however, past CBAs fail to meetthe sensitivity requirements for the quantification of pharmaceuticalpreparations of BoNT or the detection of BoNT in clinical samples.Presumably, current established model cell lines lack critical neuronalcharacteristics required for efficient BoNT uptake. A recent solution toincreasing BoNT sensitivity is the use of mouse embryonic stem cells(mESCs). Because these cells can be terminally differentiated intoneurons they are more sensitive to BoNT treatment. A major drawback tothis method, though, is the multiple weeks required to fullydifferentiate mESCs.

Thus, there is an urgent need to development cell based assays forassessing and quantifying the potency of BoNT-containing samples forbioweapon defense, food borne illness, and therapeutic purposes.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to a cultured cell that issusceptible to intoxication by a Botulinum neurotoxin. Susceptibility ofthe cell to such a neurotoxin is enhanced by contacting the cell with aprotein kinase C (PKC) inhibitor. In preferred embodiments the PKCinhibitor is an isoquinolynyl compound, for example1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride (H7). In someembodiments, inhibition of PKC with an isoquinolynyl compound such as1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride (H7),significantly enhances the intoxication of the cell by a botulinum toxinto produce a sensitized cell.

Also contemplated is optimization of a cell culture medium containingB27 for contact with such a sensitized cell and a Botulinum toxin.Essentially, the osmolarity of the culture media can be adjusted withrespect to specific Botulinum toxin preparations by modifying sodiumchloride concentration to, for example, optimize functionality withBotulinum toxin preparations that include about 15.4 mM and/orcarbohydrate concentrations to up to 0.5% and/or human serum albuminconcentrations to up to 0.1%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, and 1E are graphs showing that H7 treatment, butnot calphostin, IBMX, db-cAMP, or 8-Br-cAMP treatment, cause an increasein BoCell™ Assay sensitivity. FIG. 1A depicts the effect of H7. FIG. 1Bdepicts the effect of calphostin. FIG. 1C depicts the effect of IBMX.FIG. 1D depicts the effects of db-cAMP. FIG. 1E depicts the effects of8-Br-cAMP.

FIGS. 2A and 2B are graphs showing that the H7 effect is dose dependent,and does not affect cell viability. FIG. 2A shows the effect of H7 atdifferent concentrations on the apparent EC50 of BoNT/A neurotoxin. FIG.2B shows a histogram of cell viability at different concentrations ofH7.

FIG. 3 is a graph showing that the effect of H7 pre-treatment (beforeBoNT/A addition). Here, H7 is included with BoNT/A incubation as well.

FIGS. 4A and 4B are graphs showing that 1) only holotoxin (completeBoNT/A) but not light-chain (BoNT/A fragment) elicits a response in theBoCe11™ assay and 2) BoNT/A activity can be blocked by the addition ofHcR/A which competes for cellular receptors. Thus, BoNT/A responses inthe assay occur through natural mechanisms. FIG. 4A shows thedifferential effects of intact holotoxin BoNT/A toxin and BoNT/A toxinlight chain. FIG. 1B shows the effect of the HcR/A competitor on BoNT/Aholotoxin activity.

FIG. 5 is a graph showing that multiple H7 lots from differentmanufacturers all cause an increase in sensitivity. (There is a reportin the literature that some H7 lots from certain manufacturers are notactually H7, thus we wanted to confirm the effect with multiple lots.)

FIG. 6 is a graph showing assay response and sensitivity in the presenceof H7 following a 24 and 48 hour incubation with BoNT/A. We expect thatlimits of detection of <3 pM will be possible with assay optimization.

FIG. 7A is a schematic of a BoCell™ construct.

FIG. 7B is a schematic of an exemplary assay in which BoNT/A cell-basedreporters are used to detect BoNT/A activity by loss of YFP fluorescence

FIG. 8A is an image of BoNT/A-induced changes in fluorescence responses.

FIG. 8B is a chart showing fluorescence ratios and BoNT/A sensitivitiesof the cell-based reporters.

FIG. 8C is a blot showing activity of BoNT/A in cells regardless of thereporter.

FIG. 9 comprises multiple charts showing emission data for both YFPdegradation and loss of FRET according to the state of the art practicefrom the exact same plates of cells.

DETAILED DESCRIPTION

In preferred embodiments, the investigational substance is a Botulinumtoxin (BoNT), and the cleavage sequence is appropriately matched withthe investigational substance. For example, the BoNT/A, E, and C cleaveSNAP-25, and BoNT/B, D, F, G cleave synaptobrevin (Syb), at single butdifferent sites. BoNT/C also cleaves syntaxin in addition to SNAP-25.

Cleavage Sequence(s)

Contemplated cleavage site sequences can advantageously comprise (a) aSNARE protein, motif, or mutein. “Muteins” of a protein should beinterpreted herein as having at least 30% identity with a correspondingnative protein, including for example compositions having at least 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%identity with the native protein. Variations from identity can compriseany or more of additions, deletions and substitutions. Contemplatedmuteins include fragments, truncates and fusion proteins.

Transfected Cells

Hybrid protein(s) that are formed in the transfected cells preferablyinclude a transmembrane domain, which tends to locate to intracellularvesicles for BoNT/B, D, F, and G, and thereby present a vesicle-boundsubstrate. Heavy chain-mediated endocytosis of the BoNT into thetransfected cell is followed by presentation of the light chain on theouter surface of the vesicle, allowing the protease activity of thelight chain to cleave the cleavage sequence of the hybrid protein(s),thus cleaving the reporter-containing portion, which then is destroyedor degraded to reduce the signal being tested. Full-length Syb, forexample, contains 116 amino acids, and is localized to vesicles througha single transmembrane domain.

Hybrid protein(s) that are formed in the transfected cells preferablyinclude a transmembrane domain, which tends to locate to the plasmamembrane for BoNT/A, C, and E. In some contemplated assays, themembrane-anchoring domain comprises a fragment that contains apalmitoylation site. Suitable membrane-anchoring domains are described,for example, in US 20060134722 to Chapman.

While it is especially preferred that the transmembrane domain is thetransmembrane domain of synaptobrevin, mutations (e.g., transitions,transversions, insertions, deletions, inversions, etc.) thereof, andeven non-synaptobrevin transmembrane domains are also deemed suitablefor use herein. Similarly, it should be appreciated that thetransmembrane domain may also be replaced by another polypeptide moietythat allows at least temporary anchoring of the hybrid protein to amembrane such that the remainder of the hybrid protein is exposed to thecytosol.

With respect to the transfected cells expressing the hybrid protein itis generally preferred that the cell is stably transfected.Nevertheless, transient transfection is also contemplated. It is stillfurther typically preferred that the transfected cell is a neuronalcell. However, numerous other non-neuronal cells (including mammalian,rodent, and insect cells, and even yeast and bacterial cells) are alsocontemplated herein. Most typically, the cells will constitutivelyexpress the hybrid protein(s) are therefore under appropriate regulatoryelements. In alternative aspects, the expression may also be induced.

According to a preferred embodiment, a recombinant nucleic acidmolecule, preferably an expression vector, encoding a BoNT substratepolypeptide and a suitable reporter is introduced into a suitable hostcell. An ordinarily skilled person can choose a suitable expressionvector, preferably a mammalian expression vector, and will recognizethat there are enormous numbers of choices. For example, the pcDNAseries of vectors, such as pCI and pSi (from Promega, Madison, Wis.),CDM8, pCeo4. Many of these vectors use viral promoters. Preferably,inducible promoters are used, such as the tet-off and tet-on vectorsfrom Clontech (Mountain View, Calif.).

Many choices of cell lines are suitable as the host cell. Preferably,the cell is of a type in which the respective BoNT exhibits its toxicactivities. In other words, the cells preferably display suitable cellsurface receptors, or otherwise allow the toxin to be translocated intothe cell sufficiently efficiently, and allow the toxin to cleave thesuitable substrate polypeptide. Specific examples include primarycultured neurons (cortical neuron, hippocampal neuron, spinal cord motorneuron, etc); PC12 cells or derived PC12 cell lines; primary culturedchromaffin cells; several cultured neuroblastoma cell lines, such asmurine cholinergic Neuro 2a cell line, human adrenergic SK-N-SH cellline, and NS-26 cell line. See e.g. Foster and Stringer (1999), GeneticRegulatory Elements Introduced Into Neural Stem and Progenitor CellPopulations, Brain Pathology 9: 547-567.

The coding region for the reporter/cleavage site construct is under thecontrol of a suitable promoter. Depending on the types of host cellsused, many suitable promoters are known and readily available in theart. Such promoters can be inducible or constitutive. A constitutivepromoter may be selected to direct the expression of the desiredpolypeptide. Such an expression construct may provide additionaladvantages since it circumvents the need to culture the expression hostson a medium containing an inducing substrate. Examples of suitablepromoters would be LTR, SV40 and CMV in mammalian systems; E. coli lacor trp in bacterial systems; baculovirus polyhedron promoter (polh) ininsect systems and other promoters that are known to control expressionin eukaryotic and prokaryotic cells or their viruses. Examples of strongconstitutive and/or inducible promoters which are preferred for use infungal expression hosts are those which are obtainable from the fungalgenes for xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC),triose phosphate isomerase (tpi), alcohol dehydrogenase (AdhA),.alpha.-amylase (amy), amyloglucosidase (AG—from the glaA gene),acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd)promoters. Examples of strong yeast promoters are those obtainable fromthe genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinaseand triosephosphate isomerase. Examples of strong bacterial promotersinclude SPO2 promoters as well as promoters from extracellular proteasegenes.

Hybrid promoters may also be used to improve inducible regulation of theexpression construct. The promoter can additionally include features toensure or to increase expression in a suitable host. For example, thefeatures can be conserved regions such as a Pribnow Box or a TATA box.The promoter may even contain other sequences to affect (such as tomaintain, enhance or decrease) the levels of expression of thenucleotide sequence. For example, suitable other sequences include theShl-intron or an ADH intron. Other sequences include inducibleelements-such as temperature, chemical, light or stress inducibleelements. Also, suitable elements to enhance transcription ortranslation may be present. An example of the latter element is the TMV5′ signal sequence (see Sleat, 1987, Gene 217: 217-225; and Dawson,1993, Plant Mol. Biol. 23: 97).

The expression vector may also contain sequences which act on thepromoter to amplify expression. For example, the SV40, CMV, and polyomacis-acting elements (enhancer) and a selectable marker can provide aphenotypic trait for selection (e.g. dihydrofolate reductase or neomycinresistance for mammalian cells or amplicillin/tetracyclin resistance forE. coli). Selection of the appropriate vector containing the appropriatepromoter and selection marker is well within the level of those skilledin the art.

Preferably, the coding region for the construct is under the control ofan inducible promoter. In comparison to a constitutive promoter, aninducible promoter is preferable because it allows for suitable controlof the concentration of the reporter in the cell, therefore themeasurement of changes in signals are greatly facilitated.

For example, expression can be controlled using the Tet-on & Tet-offsystem Clontech (Mountain View, Calif.). Under the control of thispromoter, gene expression can be regulated in a precise, reversible andquantitative manner. Briefly, for Tet-on system, the transcription ofdownstream gene only happens when doxycycline is present in the culturemedium. After the transcription for a certain period of time, one canchange culture medium to deplete doxycycline, thus, stop the synthesisof new reporter proteins. Therefore, there is no background from newlysynthesized reporter proteins, and one may be able to see a fasterchange after toxin treatment.

Fluorescent Analysis

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system or afluorometer. Excitation to initiate energy emission can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range. It will be apparent tothose skilled in the art that excitation/detection means can beaugmented by the incorporation of photomultiplier means to enhancedetection sensitivity. For example, the two photon cross correlationmethod may be used to achieve the detection on a single-molecule scale(see e.g. Kohl et al., Proc. Nat'l. Acad. Sci., 99:12161, 2002).

Sensitizer

In a particular embodiment, inhibitor1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride (H7)significantly enhances the sensitivity of the BoCe11™ model cell line tobotulinum neurotoxin type A (BoNT/A).

Pretreatment of the BoCe11™ modified neuroblastoma cells with H7 causedrapid increases in both neurite length and neurite number per cells. TheH7 effect was both time and dose-dependent with maximal effects seenwith 1 mM H7 treatment. Further, this phenotypic change could be“rescued” by removal of the H7. Lastly, pretreatment of cells with H7significantly increased the sensitivity of these cells to BoNT/Atreatment as measured by SNAP-25 reporter cleavage.

H7 also inhibits cAMP and cGMP-dependent kinases, though pretreatment ofcells with the drug HA1004, a selective inhibitor of cAMP and cGMPkinases failed to induce morphologic changes in neuroblastoma cells,suggesting the effect is specific to PKC inhibition (1990, JBC paper).On the other hand, other molecules that are known neuronal celldifferentiators, including some inhibitors of PKA and PKC, do NOTincrease the sensitivity of the BoCe11™ Assay. The increased sensitivitywas not seen with other drugs previously shown to induce neuriteformation: Calphostin C, IBMX, dibutyryl-cAMP, and8-Bromoadenosine-cAMP.

It is thus contemplated that inhibition of specific PKC isoforms withselective isoquinolynyl analogues and other drugs may infer similarincreases in BoNT/A sensitivity in established model cell lines byinducing neurite formation.

It is also contemplated that the culture media can include one or bothof H7 and B27, and that the osmolarity of the culture media can bemodified to optimize effectiveness with respect to specific Botulinumtoxins, especially those in Table 1. Osmolarity can be adjusted inaccording to known principles, including especially modifying the saltand/or sugar content. As used herein, the term “optimization” meanstaking steps to improve a desired result, which may or may not actuallyachieve the best possible result.

TABLE 1 Excipient formulations of BoNT/A-based pharmaceuticals. Botox ®is manufactured by Allergan Inc. (Irvine, CA), Dysport ® by Ipsen(France), and Xeomin ® by Merz GmbH (Germany). Active Content Thera-ingredient Supplied Excipients concentrations peutic per vial form pervial in a 1 ml Botox ® 100 U Lyophilized 0.5 mg 100 U BoNT/A, BoNT/Apowder HSA, 0.9 0.05% HSA, mg NaCl 15.4 mM NaCl Dysport ®/ 500 ULyophilized 125 μg 500 U BoNT/A, Reloxin ® BoNT/A powder HSA, 2.50.0125% HSA, mg lactose 0.25% lactose Xeomin ® 100 U Lyophilized 1 mgHSA, 100 U BoNT/A, BoNT/A powder 4.7 mg 0.1% HSA, sucrose 0.47% sucrose

Sensitizer Experimental Results

Our finding is that treatment of our BoCell™ cells with the compound H7increases the sensitivity of our BoCell™ assay. From a methodsstandpoint, the possible technological advantages of H7 treatmentinclude: 1. Reduced assay times. With H7 treatment, we get BoCell™BoNT/A sensitivities after 24 hours of BoNT/A treatment that areequivalent to 72 hour BoNT/A treatments with our current assayconditions. An assay time reduction of 48 hours.

2. Overall increase in assay sensitivity. Using H7 we can increase thesensitivity of the assay by 0.5 log -1.0 log with a 48 hour BoNT/Atreatment compared to our current assay conditions. We expect thatfurther increases in sensitivity with assay optimization.

3. Increase neuronal cell sensitivity to other BoNT serotypes. We arecurrently testing this.

It is thus contemplated that use of the compounds contemplated hereincan increase the sensitivity of the assay by at least 0.5 log, at least0.6 log, at least 0.7 log, at least 0.8 log, at least 0.9 log and atleast 1.0. Unless the context dictates the contrary, all ranges setforth herein should be interpreted as being inclusive of their endpointsand open-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

FIGS. 1A to 1E, 2A, 2B, 3, 4A, 4B, 5, and 6 show data generated usingH7.

In FIGS. 1A, 1B, 1C, 1D, and 1E, BoCells were plated, and pre-treatedfor 24 hr with A) H7 (FIG. 1A), B) Calphostin C (FIG. 1B), C) IBMX (FIG.1C), D) dibutyryl-cyclic AMP (db-cAMP) (FIG. 1D), or E) 8-brom o-cyclicAMP (8-Br-cAMP) (FIG. 1E) at the indicated concentration. Control cellswere treated with vehicle equivalent to the highest drug concentrationtested. After pre-treatment, BoCells were treated with BoNT/A containingindicated drug concentration for an additional 24 hr. The assay responsewas collected by reading the emission at 485 and 535 nm using a Tecan™Infinite 500 series microplate reader. Emission ratios (YFP/CFP) wereplotted as a function of BoNT/A concentration. Data representmean±standard deviation of the mean of samples run in triplicate.

In FIGS. 2A and 2B, A) BoCells were plated and pre-treated for 24 hrwith the indicated concentration of H7. Control cells were treated withvehicle equivalent to the highest drug concentration tested. Afterpre-treatment. BoCells were treated with BoNT/A containing indicateddrug concentration for an additional 24 hr. The assay response wascollected by reading the emission at 485 and 535 nm using a Tecan™Infinite 500 series microplate reader. Emission ratios (YFP/CFP) wereplotted as a function of BoNT/A concentration (FIG. 2A). Data representmean±standard deviation of the mean of samples run in triplicate. B)BoCells were stained with trypan blue then 3× fields of 50 or more cellswere counted per treatment and cell viability calculated (FIG. 2B).

In FIG. 3, BoCells were plated and pre-treated for either 3. 6. 12 or 24hr with 0.75 mM H7. After pre-treatment. BoCells were treated withBoNT/A containing 0.75 mM H7 for an additional 24 hr. The assay responsewas collected by reading the emission at 485 and 535 nm using a TecanInfinite 500 series microplate reader. Emission ratios (YFP/CFP) wereplotted as a function of BoNT/A concentration. Data representmean±standard deviation of the mean of samples run in triplicate.

In FIGS. 4A and 4B, A) BoCells were plated and pre-treated for 24 hrwith 0.75 mM H7. After pre-treatment. BoCells were treated with eitherBoNT/A holotoxin or BoNT/A light chain containing 0.75 mM H7 for anadditional 24 hr (FIG. 4A). B.) BoCells were pre-treated for 24 hr with0.75 mM H7.10 mM HcR/A or vehicle was added to wells for 1 hr prior toaddition of BoNT/A (FIG. 4B). The assay response was collected after 24hrs by reading the emission at 485 and 535 nm using a Tecan Infinite 500series microplate reader. Emission ratios (YFP/CFP) were plotted as afunction of BoNT/A concentration. Data represent mean±standard deviationof the mean of samples run in triplicate.

In FIG. 5, BoCells were pre-incubated with 0.75 mM H7 from threeindependent manufacturers (listed) for 24 hrs then treated with BoNT/Ain the presence of 0.75 mM H7. The assay response was collected after 24hrs by reading the emission at 485 and 535 nm using a Tecan™ Infinite500 series microplate reader. Emission ratios (YFP/CFP) were plotted asa function of BoNT/A concentration. Data represent mean±standarddeviation of the mean of samples run in triplicate.

In FIG. 6, BoCells were pre-incubated with 0.75 mM H7 for 24 hrs thentreated with BoNT/A in the presence of 0.75 mM H7. The assay responsewas collected after 24 hrs and 48 hrs by reading the emission at 485 and535 nm using a Tecan™ Infinite 500 series microplate reader. Emissionratios (YFP/CFP) were plotted as a function of BoNT/A concentration.Data represent mean±standard deviation of the mean of samples run intriplicate.

Non-FRET based Assays

FIG. 7A depicts BioSentinel's BoCell™ A BoNT/A construct. The reporterfluorophore, YFP, and the normalization fluorophore, CFP, are coupled bya cleavage sequence, SNAP-25 (green). SNAP-25 palmitoylation localizesthe reporter to a plasma membrane.

FIG. 7B depicts an exemplary assay in which BoNT/A cell-based reportersare used to detect BoNT/A activity by loss of YFP fluorescence. Here,the YFP moiety is directly excited leading to fluorescence emission inthe absence of BoNT/A. Cleavage of the reporter by BoNT/A releases aC-terminal reporter fragment containing the YFP moiety into the cytosol.The fragment is rapidly degraded and, thus, YFP emission is lost. TheCFP signal is still used to control for cell-to-cell reporter expressionlevels and cell density.

Surprisingly, not all fluorescent proteins related to YFP are effectiveas the reporter fluorophore. For example, FIGS. 8A-8C provide evidencethat reporters containing YFP or the closely related derivative Venuscan detect BoNT/A activity in cells, but not mCherry or mStrawberry.Here, Neuro2A cells were grown in a 96-well plate to 70% confluency andtransiently transfected using Lipofectamine 2000 (Invitrogen™), withreporters containing the indicated N-terminal and C-terminal(N-term/C-term) fluorophore pairs. After 24 h, cells were incubated inthe presence or absence of 10 nM BoNT/A at 37° C. for 72 h in 100 μl ofphenol red-free MEM medium.

FIG. 8A shows BoNT/A-induced changes in fluorescence responses.Semi-automated YFP and CFP fluorescence measurements were performedusing a Nikon™ TE2000-U fluorescent microscope with 20× magnificationand Nikon NIS Elements 3.4 software. Shown are randomly selected fieldspseudo-colored for the C-terminal/N-terminal fluorescent protein (FP)fluorescence ratio. Ratios were calculated from emissions collected upondirect excitement of each fluorophore.

FIG. 8B represents fluorescence ratios and BoNT/A sensitivities of thecell-based reporters. 30 randomly selected cells per condition wereanalyzed for fluorescence ratios in the presence or absence of 10 nMBoNT/A. The average signal from the 30 cells from 5 microscopic fieldson 3 different wells is shown. Cells exhibiting over-saturatedfluorescence were excluded.

FIG. 8C is a blot showing that BoNT/A was active in cells regardless ofthe reporter. All reporters show some cleavage in the presence ofBoNT/A, and all native SNAP25s are cleaved. Cells were transfected andtreated with BoNT/A as described above but scaled up into 6-well plates.After 72 h incubation with BoNT/A, cells were washed 3× with serum-freeMEM, collected by scraping, and lysed using M-Per Lysis Buffer(Pierce™). 40 μg of cell lysate was subjected to SDS-PAGE beforetransfer to nitrocellulose paper and immunoblot analysis using anantibody directed against SNAP-25 (clone 71.2, Synaptic Systems). Arrowsindicate the position of the full-length (closed) and cleaved (open)forms of the reporters. Full-length (*) and cleaved (**) native SNAP-25are indicated.

The inventive subject matter can be extended beyond cleavablesubstrates, to any assay having a construct with a reporter that can bede-protected, and then degraded in some manner by the cytosol or otherlocal environment. For example, a susceptible reporter could be modifiedto include a ‘bait’ domain that is used to screen against a library ofrecombinant proteins that could possibly bind with the bait domain.Without the bait domain protected by a binding protein, the susceptiblereporter will be degraded. In such an assay, cells expressing bindingproteins will form a complex to protect the susceptible reporter fromdegradation, while cells expressing a binding partner to the bait willlight up. The bait domain could advantageously be a small peptide, andthe binding partners could be members of a library of proteins (orprotein mutants). The system could also be reversed such that there is alibrary of bait domains tested against a single test protein (or testprotein library).

In each of these instances it is considered advantageous to include asecond reporter that is not degraded post-exposure by the cytosol orother local environment, or is at least degraded much more slowlypost-exposure than the first reporter.

Still further, whereas the reporter can conveniently be selected fromsuitable fluorophores, it is contemplated that the reporter could bereplaced or augmented by any other protein or other component with adefined function that is known to (a) have a relatively fast turnover inthe cell without protection, and (b) that can be protected byinteraction with a binding partner. Defined functions includetranscription activators for reporter gene, repressors for lethal genes,etc (anything that can be easily identified or selected against).

FIG. 9 comprises multiple charts showing emission data for both YFPdegradation and loss of FRET according to the state of the art practicefrom the exact same plates of cells. For YFP degradation, directly andsingularly excited YFP emissions (top, Ex500, Em526) and CFP emissions(middle, Ex434, Em470) are collected. Those emission are then backgroundsubtracted and the YFP emission is divided by CFP emission to controlfor cell density and reporter expression in the individual wells. Thatemission ratio (YFP/CFP, bottom) is then used for the essay report.

For loss of FRET, FRET emissions (top, Ex434, Em526) and CFP emissions(middle, Ex434, Em470) are collected. Those emissions are thenbackground subtracted, and the FRET emission is divided by CFP emissionto control for cell density and reporter expression in the individualwells. That emission ratio (FRET/CFP, bottom) is shown here to compareto the normal method.

The key comparison is the loss of directly excited YFP versus the lossof FRET emission. From the comparison between the measurements and thecorresponding curves, it becomes immediately apparent that the overalldynamic range for YFP degradation is much larger than the dynamic rangeof loss of FRET emissions. In some cases, there is no difference,statistically, between cells treated with no BoNT versus sells treatedwith saturating concentrations of BoNT when looking solely at the rawFRET emissions. For the loss of FRET method, the BoNT dose response onlybecomes clear after dividing the FRET emission by the CFP (donor)emission. The CFP (donor) emission shows a small increase emission dueto de-quenching in response to reporter cleavage.

In summary, the loss of FRET method reports BoNT-induced changes in thereporter very poorly, or not at all, and therefore cannot be thereforeused for a correct qualitative and quantitative determination. Incontrast, preferred methods contemplated herein have a high degree ofspecificity and reproducibility, which allow one to rely on the data forboth the qualitative and quantitative analysis.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A sensitized cell, comprising: a cultured cellsusceptible to intoxication by a Botulinum toxin, wherein the culturedcell has been treated with a protein kinase C inhibitor, and wherein thesensitized cell is characterized by an increased susceptibility toBotulinum neurotoxin intoxication relative to the cultured cell prior totreatment with the protein kinase C inhibitor.
 2. The sensitized cell ofclaim 1, wherein the protein kinase C inhibitor is an isoquinolynylcompound.
 3. The sensitized cell of claim 2, wherein the isoquinolynylcompound is 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride(H7).
 4. The sensitized cell of claim 1, wherein the cultured cell isselected from a group consisting of a primary cultured neuron cell, PC12 cell or a derivative thereof, a primary cultured chromaphin cell, aneuroblastoma cell, a human adrenergic SK-N-SH cell, and a NS-26 cellline.
 5. The sensitized cell of claim 1, wherein the protocol comprisescontacting the cultured cell with the protein kinase C inhibitor at aconcentration of 1 mM.
 6. The sensitized cell of claim 1, wherein theprotocol comprises contacting the cultured cell with the protein kinaseC inhibitor for up to 24 hours.
 7. The sensitized cell of claim 1,wherein the cultured cell is cultured in a cell culture media optimizedfor contact with the protein kinase C inhibitor.
 8. The sensitized cellof claim 7, wherein the osmolarity of the culture media is optimized forcharacterization of a composition comprising Botulinum neurotoxin A,0.05% human serum albumin, and 15.4 mM sodium chloride.
 9. Thesensitized cell of claim 7, wherein the osmolarity of the culture mediais optimized for characterization of a composition comprising Botulinumneurotoxin A, 0.0125% human serum albumin, and 0.25% lactose.
 10. Thesensitized cell of claim 7, wherein the osmolarity of the culture mediais optimized for characterization of a composition comprising Botulinumneurotoxin A, 0.1% human serum albumin, and 0.47% sucrose.
 11. A kit forgenerating a sensitized cell, comprising: a cultured cell susceptible tointoxication by a Botulinum neurotoxin; and a protein kinase Cinhibitor.
 12. The kit of claim 11, wherein the protein kinase Cinhibitor is an isoquinolynyl compound.
 13. The kit of claim 12, whereinthe isoquinolynyl compound is1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dichloride (H7).
 14. Thekit of claim 11, wherein the cultured cell is selected from a groupconsisting of a primary cultured neuron cell, PC 12 cell or a derivativethereof, a primary cultured chromaphin cell, a neuroblastoma cell, ahuman adrenergic SK-N-SH cell, and a NS-26 cell line.
 15. The kit ofclaim 11, further comprising instructions for generating the sensitizedcell.
 16. The kit of claim 15, wherein the instructions comprise a stepof contacting the cultured cell with the protein kinase C inhibitor at aconcentration of 1 mM.
 17. The kit of claim 15, wherein the instructionscomprise a step of contacting the cultured cell with the protein kinaseC inhibitor for up to 24 hours.